Previous research had shown that there is a preferential loss of the myelin-associated glycoprotein (MAG) from the periphery of multiple sclerosis (MS) plaques in comparison to other myelin proteins including myelin basic protein (MBP), proteolipid protein (PLP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Furthermore, much of the remaining MAG in affected MS tissues is in the form of a soluble 90 kDa proteolytic cleavage product of MAG (dMAG) lacking its transmembrane and cytoplasmic domains. This proteolysis of MAG is catalyzed by a myelin- associated neutral protease that is activated in MS tissue. The soluble dMAG product was purified, and short peptides proteolytically generated from its carboxy-terminus were characterized by amino acid sequencing and mass spectroscopy. The results demonstrated that the proteolytic cleavage of MAG by the myelin-associated neutral protease occurs between residues 512 (Ala) and 513 (Lys) just outside of the membrane. The amino acid sequence around the scissile bond suggests that this proteolysis is catalyzed by a cathepsin L-like cysteine protease, and cathepsin L-like activity in myelin was confirmed by peptidolysis experiments with known cathepsin L substrates. The cathepsin L-like enzyme was purified, and amino acid sequences determined from trypsin generated peptides. Based on the peptide sequences, this putative cysteine protease was cloned and expressed in prokaryotic and eukaryotic cells. Experiments are in progress to characterize the expressed protease and study its activity on MAG. Since in adult brain MAG is localized exclusively in the periaxonal membranes of myelinating oligodendroctyes, the selective quantitative and qualitative alterations of MAG at the edge of acute MS plaques indicate that there is damage to the periaxonal membranes in this disease. These biochemical findings, as well as some morphological observations in the literature, suggest that one aspect of the pathology in some MS lesions is a dying-back oligodendrogliopathy in which the first abnormalities occur in the most distal periaxonal oligodendroglial membranes. Experiments are being undertaken in MS tissue to determine what other periaxonal abnormalities occur in this disease and to compare the results to periaxonal changes in MAG knockout mice.